Method and apparatus to fabricate polymer arrays on patterned wafers using electrochemical synthesis

ABSTRACT

A wafer having a plurality of dies (also called array chips) on the wafer, the die having an electrode to generate a deprotecting reagent, a working electrode to electrochemically synthesize a material, a confinement electrode adjacent to the working electrode to confine reactive reagents, and a die pad, wherein die pads of the plurality of dies are interconnected on the wafer to electrochemically synthesize the material in parallel on a plurality of working electrodes is disclosed. Also, a method for wafer-scale manufacturing of a plurality of dies and a method for electrochemically synthesizing a material in parallel on a plurality of dies on a wafer are disclosed.

FIELD OF INVENTION

The embodiments of the invention relate to polymer arrays prepared at awafer scale by electrochemical synthesis of a polymer on the wafer, andit relates to methods and apparatus for preparing such arrays. Theinvention transcends several scientific disciplines such as polymerchemistry, biochemistry, molecular biology, medicine and medicaldiagnostics.

BACKGROUND

Synthesis of high density polymer arrays on a microarray chip is known.Examples of such high density polymer arrays include nucleic acidarrays, peptide arrays, and carbohydrate arrays.

One method of preparing polymer arrays on microarray chips involvesphotolithographic techniques using photocleavable protecting groups.Briefly, the method includes attaching photoreactive groups to thesurface of a substrate, exposing selected regions of the substrate tolight to activate those regions, attaching a monomer with aphotoremovable group to the activated regions, and repeating the stepsof activation and attachment until macromolecules of the desired lengthand sequence are synthesized.

Additional methods and techniques applicable to polymer array synthesisinclude electrochemical synthesis. One example includes providing aporous substrate with an electrode therein, placing a molecule having aprotected chemical group in proximity of the porous substrate, placing abuffering solution in contact with the electrode and the poroussubstrate to prevent electrochemically generated reagents from leavingthe locality of the electrode (the use of confinement electrodes toprevent reagents from diffusing away have also been described), applyinga potential to the electrode to generate electrochemical reagentscapable of deprotecting the protected chemical functional group of themolecule, attaching the deprotected chemical functional group to theporous substrate or a molecule on the substrate, and repeating the abovesteps until polymers of the desired length and sequence are synthesized.

The above-mentioned functionalization methods are carried out onepolymer array chip at a time, resulting in high unit cost of the polymerarray chip due to the resulting limitations on manufacturingscalability. For many major applications of interest, such high unitcost is likely to be prohibitive (e.g., for disease diagnostics based onthe use of high volumes of DNA arrays). The embodiments of the inventionmeet this need.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a) shows the layout of a wafer with pads on the dies (DP1, DP2,DP3) interconnected across the scribe lines to the contact pads on thewafer (WP1, WP2, WP3). FIG. 1( b) shows a portion of electrode array ona die called an element, pad, or spot containing anode, working andconfinement electrodes. FIG. 1( c) shows a portion of the wafer withinterconnects to the backside of the wafer.

FIG. 2 shows a CMOS switching scheme for individually addressableelectrodes.

FIG. 3 shows an acid-catalyzed polymer synthesis scheme of an embodimentof the invention.

DETAILED DESCRIPTION

Nucleic acids (DNA and RNA) can form double-stranded molecules byhybridization, that is, complementary base pairing. The specificity ofnucleic acid hybridization is such that a particular DNA or RNA moleculecan be labeled (e.g., with a radioactive or fluorescent tag) to generatea probe, and be used to isolate a complementary molecule (target) from avery complex mixture, such as a whole genomic DNA or whole cellular RNA.It is also possible to label the target instead of labeling the probe orlabel both the probe and the target. This specificity of complementarybase pairing also allows thousands of hybridization to be carried outsimultaneously in the same experiment on a DNA chip; (also called a DNAarray).

The polymer arrays of the embodiment of the invention could be a DNAarray (collections of DNA probes on a shared base) comprising a densegrid of spots (often called elements or pads) arranged on a miniaturesupport. Each spot could represent a different gene.

The probe in a DNA chip is usually hybridized with a complex RNA or cDNAtarget generated by making labeled DNA copies of a complex mixture ofRNA molecules derived from a particular cell type (source). Thecomposition of such a target reflects the level of individual RNAmolecules in the source. The intensities of the signals from the DNAspots of the DNA chip after hybridization between the probe and thetarget represent the relative expression levels of the genes of thesource.

The DNA chip could be used for differential gene expression betweensamples (e.g., healthy tissue versus diseased tissue) to search forvarious specific genes (e.g., connected with an infectious agent) or ingene polymorphism and expression analysis. Particularly, the DNA chipcould be used to investigate expression of various genes connected withvarious diseases in order to find causes of these diseases and to enableaccurate treatments.

Using an embodiment of the polymer array of the invention, one couldfind a specific segment of a nucleic acid of a gene, i.e., find a sitewith a particular order of bases in the examined gene. This detectioncould be performed by using a diagnostic oligonucleotide made up ofshort synthetically assembled single-chained complementaryoligonucleotide—a chain of bases organized in a mirror order to whichthe specific segment of the nucleic acid would attach (hybridize) viaA-T or G-C bonds.

The practice of the embodiments of the invention may employ, unlessotherwise indicated, conventional techniques of organic chemistry,polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an array” may include a plurality ofarrays unless the context clearly dictates otherwise.

An “array” is an intentionally created collection of molecules which canbe prepared either synthetically or biosynthetically. The molecules inthe array can be identical or different from each other. The array canassume a variety of formats, e.g., libraries of soluble molecules;libraries of compounds tethered to resin beads, silica chips, or othersolid supports. The array could either be a macroarray or a microarray,depending on the size of the sample spots on the array. A macroarraygenerally contains sample spot sizes of about 300 microns or larger andcan be easily imaged by gel and blot scanners. A microarray wouldgenerally contain spot sizes of less than 300 microns.

“Solid support,” “support,” and “substrate” refer to a material or groupof materials having a rigid or semi-rigid surface or surfaces. In someaspects, at least one surface of the solid support will be substantiallyflat, although in some aspects it may be desirable to physicallyseparate synthesis regions for different molecules with, for example,wells, raised regions, pins, etched trenches, or the like. In certainaspects, the solid support(s) will take the form of beads, resins, gels,microspheres, or other geometric configurations.

The term “probe” refers to the diagnostic oligonucleotide, which istypically a fluorescently labeled DNA or RNA.

The term “target” refers to a molecule attached to the substrate of thearray, which is typically cDNA or pre-synthesized oligonucleotidedeposited on the array. The oligonucleotide targets require only thesequence information of genes, and thereby can exploit the genomesequences of an organism. In cDNA arrays, there could becross-hybridization due to sequence homologies among members of a genefamily. Oligonucleotide arrays can be specifically designed todifferentiate between highly homologous members of a gene family as wellas spliced forms of the same gene (exon-specific). Oligonucleotidearrays of the embodiment of this invention could also be designed toallow detection of mutations and single nucleotide polymorphism.

The terms “die,” “polymer array chip,” “DNA array,” “array chip,” “DNAarray chip,” “bio-chip” or “chip” are used interchangeably and refer toa collection of a large number of targets arranged on a shared substratewhich could be a portion of a silicon wafer, a nylon strip or a glassslide.

The term “molecule” generally refers to a macromolecule or polymer asdescribed herein. However, arrays comprising single molecules, asopposed to macromolecules or polymers, are also within the scope of theembodiments of the invention.

“Predefined region” or “spot” or “pad” refers to a localized area on asolid support which is, was, or is intended to be used for formation ofa selected molecule and is otherwise referred to herein in thealternative as a “selected” region. The predefined region may have anyconvenient shape, e.g., circular, rectangular, elliptical, wedge-shaped,etc. For the sake of brevity herein, “predefined regions” are sometimesreferred to simply as “regions” or “spots.” In some embodiments, apredefined region and, therefore, the area upon which each distinctmolecule is synthesized is smaller than about 1 cm² or less than 1 mm²,and still more preferably less than 0.5 mm². In most preferredembodiments the regions have an area less than about 10,000 μm² or, morepreferably, less than 100 μm². Additionally, multiple copies of thepolymer will typically be synthesized within any preselected region. Thenumber of copies can be in the thousands to the millions. Morepreferably, a die of a wafer contains at least 400 spots in, forexample, an at least 20×20 matrix. Even more preferably, the diecontains at least 2048 spots in, for example, an at least 64×32 matrix,and still more preferably, the die contains at least 204,800 spots in,for example, an at least 640×320 array.

A spot could contain an electrode to generate an electrochemicalreagent, a working electrode to synthesize a polymer and a confinementelectrode to confine the generated electrochemical reagent. Theelectrode to generate the electrochemical reagent could be of any shape,including, for example, circular, flat disk shaped and hemisphereshaped.

The electrode to generate an electrochemical reagent, the workingelectrode or the confinement electrode that may be used in embodimentsof the invention may be composed of, but are not limited to, metals suchas iridium and/or platinum, and other metals, such as, palladium, gold,silver, copper, mercury, nickel, zinc, titanium, tungsten, aluminum, aswell as alloys of various metals, and other conducting materials, suchas, carbon, including glassy carbon, reticulated vitreous carbon, basalplane graphite, edge plane graphite and graphite. Doped oxides such asindium tin oxide, and semiconductors such as silicon oxide and galliumarsenide are also contemplated. Additionally, the electrodes may becomposed of conducting polymers, metal doped polymers, conductingceramics and conducting clays. Among the metals, platinum and palladiumare especially preferred because of the advantageous propertiesassociated with their ability to absorb hydrogen, i.e., their ability tobe “preloaded” with hydrogen before being used in the methods of theinvention.

The electrode(s) may be connected to an electric source in any knownmanner. Preferred ways of connecting the electrodes to the electricsource include CMOS (complementary metal oxide semiconductor) switchingcircuitry, radio and microwave frequency addressable switches, lightaddressable switches, direct connection from an electrode to a bond padon the perimeter of a semiconductor chip, and combinations thereof. CMOSswitching circuitry involves the connection of each of the electrodes toa CMOS transistor switch. The switch could be accessed by sending anelectronic address signal down a common bus to SRAM (static randomaccess memory) circuitry associated with each electrode. When the switchis “on”, the electrode is connected to an electric source. Radio andmicrowave frequency addressable switches involve the electrodes beingswitched by a RF or microwave signal. This allows the switches to bethrown both with and/or without using switching logic. The switches canbe tuned to receive a particular frequency or modulation frequency andswitch without switching logic. Light addressable switches are switchedby light. In this method, the electrodes can also be switched with andwithout switching logic. The light signal can be spatially localized toafford switching without switching logic. This could be accomplished,for example, by scanning a laser beam over the electrode array; theelectrode being switched each time the laser illuminates it.

In some aspects, a predefined region can be achieved by physicallyseparating the regions (i.e., beads, resins, gels, etc.) into wells,trays, etc.

A “protecting group” is a moiety which is bound to a molecule anddesigned to block one reactive site in a molecule, but may be spatiallyremoved upon selective exposure to an activator or a deprotectingreagent. Several examples of protecting groups are known in theliterature. The proper selection of protecting group (also known asprotective group) for a particular synthesis would be governed by theoverall methods employed in the synthesis. Activators include, forexample, electromagnetic radiation ion beams, electric fields, magneticfields, electron beams, x-ray, and the like. A deprotecting reagentcould include, for example, an acid, a base or a free radical.

Protective groups are materials that bind to a monomer, a linkermolecule or a pre-formed molecule to protect a reactive functionality onthe monomer, linker molecule or pre-formed molecule, which may beremoved upon selective exposure to an activator, such as anelectrochemically generated reagent. Protective groups that may be usedin accordance with an embodiment of the invention preferably include allacid and base labile protecting groups. For example, peptide aminegroups are preferably protected by t-butyloxycarbonyl (BOC) orbenzyloxycarbonyl (CBZ), both of which are acid labile, or by9-fluorenylmethoxycarbonyl (FMOC), which is base labile. Additionally,hydroxyl groups on phosphoramidites may be protected by dimethoxytrityl(DMT), which is acid labile. Exocyclic amine groups on nucleosides, inparticular on phosphoramidites, are preferably protected bydimethylformamidine on the adenosine and guanosine bases, and isobutyrylon the cytidine bases, both of which are base labile protecting groups.This protection strategy is known as fast oligonucleotide deprotection(FOD). Phosphoramidites protected in this manner are known as FODphosphoramidites.

Any unreacted deprotected chemical functional groups may be capped atany point during a synthesis reaction to avoid or to prevent furtherbonding at such molecule. Capping groups “cap” deprotected functionalgroups by, for example, binding with the unreacted amino functions toform amides. Capping agents suitable for use in an embodiment of theinvention include: acetic anhydride, n-acetylimidizole, isopropenylformate, fluorescamine, 3-nitrophthalic anhydride and 3-sulfoproponicanhydride. Of these, acetic anhydride and n-acetylimidizole arepreferred.

Additional protecting groups that may be used in accordance with anembodiment of the invention include acid labile groups for protectingamino moieties: tertbutyloxycarbonyl,- tert-amyloxycarbonyl,adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl,2-(p-biphenyl)propyl(2)oxycarbonyl,2-(p-phenylazophenylyl)propyl(2)oxycarbonyl,alpha.,.alpha.-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl,2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl,benzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl (trityl),p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl,diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl,and 1-naphthylidene; as base labile groups for protecting aminomoieties: 9-fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl,and 5-benzisoazolylmethyleneoxycarbonyl; as groups for protecting aminomoieties that are labile when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; as groups for protecting aminomoieties that are labile when oxidized: (ethylthio)carbonyl; as groupsfor protecting amino moieties that are labile to miscellaneous reagents,the appropriate agent is listed in parenthesis after the group:phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl(2-aminothiophenol); acid labile groups for protecting carboxylic acids:tert-butyl ester; acid labile groups for protecting hydroxyl groups:dimethyltrityl; and basic labile groups for protecting phosphotriestergroups: cyanoethyl.

The term “electrochemical” refers to an interaction or interconversionof electric and chemical phenomena.

An “electrochemical reagent” refers to a chemical generated at aselected electrode by applying a sufficient electrical potential to theselected electrode and is capable of electrochemically removing aprotecting group from a chemical functional group. The chemical groupwould generally be attached to a molecule. Removal of a protectinggroup, or “deprotection,” in accordance with the invention, preferablyoccurs at a particular portion of a molecule when a chemical reagentgenerated by the electrode acts to deprotect or remove, for example, anacid or base labile protecting group from the molecule. Thiselectrochemical deprotection reaction may be direct, or may involve oneor more intermediate chemical reactions that are ultimately driven orcontrolled by the imposition of sufficient electrical potential at aselected electrode.

Electrochemical reagents that can be generated electrochemically at anelectrode fall into two broad classes: oxidants and reductants. Oxidantsthat can be generated electrochemically, for example, include iodine,iodate, periodic acid, hydrogen peroxide, hypochlorite, metavanadate,bromate, dichromate, cerium (IV), and permanganate ions. Reductants thatcan be generated electrochemically, for example, include chromium (II),ferrocyanide, thiols, thiosulfate, titanium (III), arsenic (III) andiron (II) ions. The miscellaneous reagents include bromine, chloride,protons and hydroxyl ions. Among the foregoing reagents, protons,hydroxyl, iodine, bromine, chlorine and thiol ions are preferred.

The generation of and electrochemical reagent of a desired type ofchemical species requires that the electric potential of the electrodethat generates the electrochemical reagent have a certain value, whichmay be achieved by specifying either the voltage or the current. Thereare two ways to achieve the desired potential at this electrode: eitherthe voltage may be specified at a desired value or the current can bedetermined such that it is sufficient to provide the desired voltage.The range between the minimum and maximum potential values could bedetermined by the type of electrochemical reagent chosen to begenerated.

An “activating group” refers to those groups which, when attached to aparticular chemical functional group or reactive site, render that sitemore reactive toward covalent bond formation with a second chemicalfunctional group or reactive site.

A “polymeric brush” ordinarily refers to polymer films comprising chainsof polymers that are attached to the surface of a substrate. Thepolymeric brush could be a functionalized polymer films which comprisefunctional groups such as hydroxyl, amino, carboxyl, thiol, amide,cyanate, thiocyanate, isocyanate and isothio cyanate groups, or acombination thereof, on the polymer chains at one or more predefinedregions. The polymeric brushes of the embodiment of the invention arecapable of attachment or stepwise synthesis of macromolecules thereon.

A “linker” molecule refers to any of those molecules described supra andpreferably should be about 4 to about 40 atoms long to providesufficient exposure. The linker molecules may be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units,diamines, diacids, amino acids, among others, and combinations thereof.Alternatively, the linkers may be the same molecule type as that beingsynthesized (i.e., nascent polymers), such as polynucleotides,oligopeptides, or oligosaccharides.

The linker molecule or substrate itself and monomers used herein areprovided with a functional group to which is bound a protective group.Generally, the protective group is on the distal or terminal end of amolecule. Preferably, the protective group is on the distal or terminalend of the linker molecule opposite the substrate. The protective groupmay be either a negative protective group (i.e., the protective grouprenders the linker molecules less reactive with a monomer upon exposure)or a positive protective group (i.e., the protective group renders thelinker molecules more reactive with a monomer upon exposure). In thecase of negative protective groups an additional step of reactivationwill be required. In some embodiments, this will be done by heating.

The polymer brush or the linker molecule may be provided with acleavable group at an intermediate position, which group can be cleavedwith an electrochemically generated reagent. This group is preferablycleaved with a reagent different from the reagent(s) used to remove theprotective groups. This enables removal of the various synthesizedpolymers or nucleic acid sequences following completion of thesynthesis. The cleavable group could be acetic anhydride,n-acetylimidizole, isopropenyl formate, fluorescamine, 3-nitrophthalicanhydride and 3-sulfoproponic anhydride. Of these, acetic anhydride andn-acetylimidizole are preferred.

The polymer brush or the linker molecule could be of sufficient lengthto permit polymers on a completed substrate to interact freely withbinding entities (monomers, for example) exposed to the substrate. Thepolymer brush or the linker molecule, when used, could preferably belong enough to provide sufficient exposure of the functional groups tothe binding entity. The linker molecules may include, for example, arylacetylene, ethylene glycol oligomers containing from 2 to 20 monomerunits, diamines, diacids, amino acids, and combinations thereof. Otherlinker molecules may be used in accordance with the differentembodiments of the present invention and will be recognized by thoseskilled in the art in light of this disclosure. In one embodiment,derivatives of the acid labile 4,4′-dimethyoxytrityl molecules with anexocyclic active ester can be used in accordance with an embodiment ofthe invention. More preferably,N-succinimidyl-4[bis-(4-methoxyphenyl)-chloromethyl]-benzoate is used asa cleavable linker molecule during DNA synthesis. Alternatively, othermanners of cleaving can be used over the entire array at the same time,such as chemical reagents, light or heat.

A “free radical initiator” or “initiator” is a compound that can providea free radical under certain conditions such as heat, light, or otherelectromagnetic radiation, which free radical can be transferred fromone monomer to another and thus propagate a chain of reactions throughwhich a polymer may be formed. Several free radical initiators are knownin the art, such as azo, nitroxide, and peroxide types, or thosecomprising multi-component systems.

“Living free radical polymerization” is defined as a livingpolymerization process wherein chain initiation and chain propagationoccur without significant chain termination reactions. Each initiatormolecule produces a growing monomer chain which continuously propagatesuntil all the available monomer has been reacted. Living free radicalpolymerization differs from conventional free radical polymerizationwhere chain initiation, chain propagation and chain terminationreactions occur simultaneously and polymerization continues until theinitiator is consumed. Living free radical polymerization facilitatescontrol of molecular weight and molecular weight distribution. Livingfree radical polymerization techniques, for example, involve reversibleend capping of growing chains during polymerization. One example is atomtransfer radical polymerization (ATRP).

A “radical generation site” is a site on an initiator wherein freeradicals are produced in response to heat or electromagnetic radiation.

A “polymerization terminator” is a compound that prevents a polymerchain from further polymerization. These compounds may also be known as“terminators,” or “capping agents” or “inhibitors.” Variouspolymerization terminators are known in the art. In one aspect, amonomer that has no free hydroxyl groups may act as a polymerizationterminator.

The term “capable of supporting polymer array synthesis” refers to anybody on which polymer array synthesis can be carried out, e.g., apolymeric brush that is functionalized with functional groups such ashydroxyl, amino, carboxyl etc. These functional groups permitmacromolecular synthesis by acting as “attachment points.”

The monomers in a given polymer or macromolecule can be identical to ordifferent from each other. A monomer can be a small or a large molecule,regardless of molecular weight. Furthermore, each of the monomers may beprotected members which are modified after synthesis.

“Monomer” as used herein refers to those monomers that are used to aform a polymer. However, the meaning of the monomer will be clear fromthe context in which it is used. The monomers for forming the polymersof the embodiments of the invention, e.g., a polymeric brush or a linkermolecule, have for example the general structure:

wherein R¹ is hydrogen or lower alkyl; R₂ and R₃ are independentlyhydrogen, or —Y—Z, wherein Y is lower alkyl, and Z is hydroxyl, amino,or C(O)—R, where R is hydrogen, lower alkoxy or aryloxy.

The term “alkyl” refers to those groups such as methyl, ethyl, propyl,butyl etc, which may be linear, branched or cyclic.

The term “alkoxy” refers to groups such as methoxy, ethoxy, propoxy,butoxy, etc., which may be linear, branched or cyclic.

The term “lower” as used in the context of lower alkyl or lower alkoxyrefers to groups having one to six carbons.

The term “aryl” refers to an aromatic hydrocarbon ring to which isattached an alkyl group. The term “aryloxy” refers to an aromatichydrocarbon ring to which is attached an alkoxy group. One of ordinaryskill in the art would readily understand these terms.

Other monomers for preparing macromolecules of the embodiments of theinvention are well-known in the art. For example, when the macromoleculeis a peptide, the monomers include, but are not restricted to, forexample, amino acids such as the L-amino acids, the D-amino acids, thesynthetic and/or natural amino acids. When the macromolecule is anucleic acid, or polynucleotide, the monomers include any nucleotide.When the macromolecule is a polysaccharide, the monomers can be anypentose, hexose, heptose, or their derivatives.

A “monomer addition cycle” is a cycle comprising the chemical reactionsnecessary to produce covalent attachment of a monomer to a nascentpolymer or linker, such as to elongate the polymer with the desiredchemical bond (e.g., 5′-3′ phosphodiester bond, peptide bond, etc.). Forexample, and not to limit the invention, the following steps typicallycomprise a monomer addition cycle in phosphoramidite-basedpolynucleotide synthesis: (1) deprotection, comprising removal of theDMT group from a 5′-protected nucleoside (which may be part of a nascentpolynucleotide) wherein the 5′-hydroxyl is blocked by covalentattachment of DMT, such deprotection is usually done with a suitabledeprotection reagent (e.g., a protic acid: trichloroacetic acid ordichloroacetic acid), and may include physical removal (e.g., washing,such as with acetonitrile) of the removed protecting group (e.g., thecleaved dimethyltrityl group), (2) coupling, comprising reacting aphosphoramidite nucleoside(s), often activated with tetrazole, with thedeprotected nucleoside, (3) optionally including capping, to truncateunreacted nucleosides from further participation in subsequent monomeraddition cycles, such as by reaction with acetic anhydride andN-methylimidazole to acetylate free 5′-hydroxyl groups, and (4)oxidation, such as by iodine in tetrahydrofuran/water/pyridine, toconvert the trivalent phosphite triester linkage to a pentavalentphosphite triester, which in turn can be converted to a phosphodiestervia reaction with ammonium hydroxide. Thus, with respect tophosphoramidite synthesis of polynucleotides, the following reagents aretypically necessary for a complete monomer addition cycle:trichloroacetic acid or dichloroacetic acid, a phosphoramiditenucleoside, an oxidizing agent, such as iodine (e.g.,iodine/water/THF/pyridine), and optionally N-methylimidazole forcapping.

A “macromolecule” or “polymer” comprises two or more monomers covalentlyjoined. The monomers may be joined one at a time or in strings ofmultiple monomers, ordinarily known as “oligomers.” Thus, for example,one monomer and a string of five monomers may be joined to form amacromolecule or polymer of six monomers. Similarly, a string of fiftymonomers may be joined with a string of hundred monomers to form amacromolecule or polymer of one hundred and fifty monomers. The termpolymer as used herein includes, for example, both linear and cyclicpolymers of nucleic acids, polynucleotides, polynucleotides,polysaccharides, oligosaccharides, proteins, polypeptides, peptides,phospholipids and peptide nucleic acids (PNAs). The peptides includethose peptides having either α-, β-, or ω-amino acids. In addition,polymers include heteropolymers in which a known drug is covalentlybound to any of the above, polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, polyacetates, or other polymers which will beapparent upon review of this disclosure.

A “nano-material” as used herein refers to a structure, a device or asystem having a dimension at the atomic, molecular or macromolecularlevels, in the length scale of approximately 1-100 nanometer range.Preferably, a nano-material has properties and functions because of thesize and can be manipulated and controlled on the atomic level.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides, that comprise purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. Polynucleotides of the embodiments of theinvention include sequences of deoxyribopolynucleotide (DNA),ribopolynucleotide (RNA), or DNA copies of ribopolynucleotide (cDNA)which may be isolated from natural sources, recombinantly produced, orartificially synthesized. A further example of a polynucleotide of theembodiments of the invention may be polyamide polynucleotide (PNA). Thepolynucleotides and nucleic acids may exist as single-stranded ordouble-stranded. The backbone of the polynucleotide can comprise sugarsand phosphate groups, as may typically be found in RNA or DNA, ormodified or substituted sugar or phosphate groups. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. The polymers made of nucleotides such asnucleic acids, polynucleotides and polynucleotides may also be referredto herein as “nucleotide polymers.

The term “nucleotide” includes deoxynucleotides and analogs thereof.These analogs are those molecules having some structural features incommon with a naturally occurring nucleotide such that when incorporatedinto a polynucleotide sequence, they allow hybridization with acomplementary polynucleotide in solution. Typically, these analogs arederived from naturally occurring nucleotides by replacing and/ormodifying the base, the ribose or the phosphodiester moiety. The changescan be tailor-made to stabilize or destabilize hybrid formation, or toenhance the specificity of hybridization with a complementarypolynucleotide sequence as desired, or to enhance stability of thepolynucleotide.

Analogs also include protected and/or modified monomers as areconventionally used in polynucleotide synthesis. As one of skill in theart is well aware, polynucleotide synthesis uses a variety ofbase-protected nucleoside derivatives in which one or more of thenitrogens of the purine and pyrimidine moiety are protected by groupssuch as dimethoxytrityl, benzyl, tert-butyl, isobutyl and the like.

For instance, structural groups are optionally added to the ribose orbase of a nucleoside for incorporation into a polynucleotide, such as amethyl, propyl or allyl group at the 2′-O position on the ribose, or afluoro group which substitutes for the 2′-O group, or a bromo group onthe ribonucleoside base. 2′-O-methyloligoribonucleotides (2′-O-MeORNs)have a higher affinity for complementary polynucleotides (especiallyRNA) than their unmodified counterparts. Alternatively, deazapurines anddeazapyrimidines in which one or more N atoms of the purine orpyrimidine heterocyclic ring are replaced by C atoms can also be used.

The phosphodiester linkage, or “sugar-phosphate backbone” of thepolynucleotide can also be substituted or modified, for instance withmethyl phosphonates, O-methyl phosphates or phosphororthioates. Anotherexample of a polynucleotide comprising such modified linkages forpurposes of this disclosure includes “peptide polynucleotides” in whicha polyamide backbone is attached to polynucleotide bases, or modifiedpolynucleotide bases. Peptide polynucleotides which comprise a polyamidebackbone and the bases found in naturally occurring nucleotides arecommercially available.

Nucleotides with modified bases can also be used in the embodiments ofthe invention. Some examples of base modifications include2-aminoadenine, 5-methylcytosine, 5-(propyn-1-yl)cytosine,5-(propyn-1-yl)uracil, 5-bromouracil, 5-bromocytosine,hydroxymethylcytosine, methyluracil, hydroxymethyluracil, anddihydroxypentyluracil which can be incorporated into polynucleotides inorder to modify binding affinity for complementary polynucleotides.

Groups can also be linked to various positions on the nucleoside sugarring or on the purine or pyrimidine rings which may stabilize the duplexby electrostatic interactions with the negatively charged phosphatebackbone, or through interactions in the major and minor groves. Forexample, adenosine and guanosine nucleotides can be substituted at theN² position with an imidazolyl propyl group, increasing duplexstability. Universal base analogues such as 3-nitropyrrole and5-nitroindole can also be included. A variety of modifiedpolynucleotides suitable for use in the embodiments of the invention aredescribed in the literature.

When the macromolecule of interest is a peptide, the amino acids can beany amino acids, including α, β, or ω-amino acids. When the amino acidsare α-amino acids, either the L-optical isomer or the D-optical isomermay be used. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also contemplated by theembodiments of the invention. These amino acids are well-known in theart.

A “peptide” is a polymer in which the monomers are amino acids and whichare joined together through amide bonds and alternatively referred to asa polypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are two or more amino acid monomers long, andoften more than 20 amino acid monomers long.

A “protein” is a long polymer of amino acids linked via peptide bondsand which may be composed of two or more polypeptide chains. Morespecifically, the term “protein” refers to a molecule composed of one ormore chains of amino acids in a specific order; for example, the orderas determined by the base sequence of nucleotides in the gene coding forthe protein. Proteins are required for the structure, function, andregulation of the body's cells, tissues, and organs, and each proteinhas unique functions. Examples are hormones, enzymes, and antibodies.

The term “sequence” refers to the particular ordering of monomers withina macromolecule and it may be referred to herein as the sequence of themacromolecule.

The term “hybridization” refers to the process in which twosingle-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” For example, hybridization refers to theformation of hybrids between a probe polynucleotide (e.g., apolynucleotide of the invention which may include substitutions,deletion, and/or additions) and a specific target polynucleotide (e.g.,an analyte polynucleotide) wherein the probe preferentially hybridizesto the specific target polynucleotide and substantially does nothybridize to polynucleotides consisting of sequences which are notsubstantially complementary to the target polynucleotide. However, itwill be recognized by those of skill that the minimum length of apolynucleotide required for specific hybridization to a targetpolynucleotide will depend on several factors: G/C content, positioningof mismatched bases (if any), degree of uniqueness of the sequence ascompared to the population of target polynucleotides, and chemicalnature of the polynucleotide (e.g., methylphosphonate backbone,phosphorothiolate, etc.), among others.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known in the art.

It is appreciated that the ability of two single strandedpolynucleotides to hybridize will depend upon factors such as theirdegree of complementarity as well as the stringency of the hybridizationreaction conditions.

As used herein, “stringency” refers to the conditions of a hybridizationreaction that influence the degree to which polynucleotides hybridize.Stringent conditions can be selected that allow polynucleotide duplexesto be distinguished based on their degree of mismatch. High stringencyis correlated with a lower probability for the formation of a duplexcontaining mismatched bases. Thus, the higher the stringency, thegreater the probability that two single-stranded polynucleotides,capable of forming a mismatched duplex, will remain single-stranded.Conversely, at lower stringency, the probability of formation of amismatched duplex is increased.

The appropriate stringency that will allow selection of aperfectly-matched duplex, compared to a duplex containing one or moremismatches (or that will allow selection of a particular mismatchedduplex compared to a duplex with a higher degree of mismatch) isgenerally determined empirically. Means for adjusting the stringency ofa hybridization reaction are well-known to those of skill in the art.

A “ligand” is a molecule that is recognized by a particular receptor.Examples of ligands that can be investigated by this invention include,but are not restricted to, agonists and antagonists for cell membranereceptors, toxins and venoms, viral epitopes, hormones, hormonereceptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g.opiates, steroids, etc.), lectins, sugars, polynucleotides, nucleicacids, oligosaccharides, proteins, and monoclonal antibodies.

A “receptor” is molecule that has an affinity for a given ligand.Receptors may-be naturally-occurring or manmade molecules. Also, theycan be employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the termreceptors is used herein, no difference in meaning is intended. A“Ligand Receptor Pair” is formed when two macromolecules have combinedthrough molecular recognition to form a complex. Other examples ofreceptors which can be investigated by this invention include but arenot restricted to:

-   -   a) Microorganism receptors: Determination of ligands which bind        to receptors, such as specific transport proteins or enzymes        essential to survival of microorganisms, is useful in developing        a new class of antibiotics. Of particular value would be        antibiotics against opportunistic fungi, protozoa, and those        bacteria resistant to the antibiotics in current use.    -   b) Enzymes: For instance, one type of receptor is the binding        site of enzymes such as the enzymes responsible for cleaving        neurotransmitters; determination of ligands which bind to        certain receptors to modulate the action of the enzymes which        cleave the different neurotransmitters is useful in the        development of drugs which can be used in the treatment of        disorders of neurotransmission.    -   c) Antibodies: For instance, the invention may be useful in        investigating the ligand-binding site on the antibody molecule        which combines with the epitope of an antigen of interest;        determining a sequence that mimics an antigenic epitope may lead        to the development of vaccines of which the immunogen is based        on one or more of such sequences or lead to the development of        related diagnostic agents or compounds useful in therapeutic        treatments such as for auto-immune diseases (e.g., by blocking        the binding of the “anti-self” antibodies).    -   d) Nucleic Acids: Sequences of nucleic acids may be synthesized        to establish DNA or RNA binding sequences.    -   e) Catalytic Polypeptides: Polymers, preferably polypeptides,        which are capable of promoting a chemical reaction involving the        conversion of one or more reactants to one or more products.        Such polypeptides generally include a binding site specific for        at least one reactant or reaction intermediate and an active        functionality proximate to the binding site, which functionality        is capable of chemically modifying the bound reactant.    -   f) Hormone receptors: Examples of hormones receptors include,        e.g., the receptors for insulin and growth hormone.        Determination of the ligands which bind with high affinity to a        receptor is useful in the development of, for example, an oral        replacement of the daily injections which diabetics must take to        relieve the symptoms of diabetes. Other examples are the        vasoconstrictive hormone receptors; determination of those        ligands which bind to a receptor may lead to the development of        drugs to control blood pressure.    -   g) Opiate receptors: Determination of ligands which bind to the        opiate receptors in the brain is useful in the development of        less-addictive replacements for morphine and related drugs.

The term “complementary” refers to the topological compatibility ormatching together of interacting surfaces of a ligand molecule and itsreceptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

A “probe card” refers to a card whose conducting components are isolatedfrom the environment (for example, the fluid reagents). All conductingsurfaces should preferably be isolated—including the pad and point ofconnection between the pad and probe.

A “scribe line” is typically an “inactive” area between the active diesthat provide area for separating the die (usually with a saw). Often,metrology and alignment features populate this area.

A “via interconnection” refers to a hole etched in the interlayer of adielectric which is then filled with an electrically conductivematerial, preferably tungsten, to provide vertical electrical connectionbetween stacked up interconnect metal lines that are capable ofconducting electricity.

“Metal lines” within a die are interconnect lines. Unlike in theembodiments of this invention, metal interconnect lines do not typicallycross the scribe line boundary to electrically connect two dies or, asin the some embodiments of this invention, a multitude of die to one ormore wafer pads.

The term “oxidation” means losing electron to oxidize. The term“reduction” means gaining electrons to reduce. The term “redox reaction”refers to any chemical reaction which involves oxidation and reduction.

The term “wafer” means a semiconductor substrate. A wafer could befashioned into various sizes and shapes. It could be used as a substratefor a microchip. The substrate could be overlaid or embedded withcircuitry, for example, a pad, via, an interconnect or a scribe line.The circuitry of the wafer could also serve several purpose, forexample, as microprocessors, memory storage, and/or communicationcapabilities. The circuitry can be controlled by the microprocessor onthe wafer itself or controlled by a device external to the wafer.

The embodiments of the invention are directed to placing molecules inparallel on a plurality of predefined regions on a substrate such as asilicon wafer or a material on a silicon wafer, wherein the moleculesare selected generally from monomers, linker molecules, polymer brush,and pre-formed molecules, including, in particular, nucleic acids. Theembodiments of the invention are more particularly directed to thesynthesis of polymers in parallel at a plurality of predefined regionson a substrate, and in particular polypeptides, by means of apolymerization technique, which generally involves the electrochemicalremoval of a protecting group from a molecule that is proximate anelectrode. The embodiments of the invention are also particularlydirected to the synthesis of oligonucleotides and/or DNA in parallel atselected predefined regions on a substrate, by means of the disclosedpolymerization technique.

However, first consider the following problem recognized by theinventors in the current electrochemistry-based array functionalizationmethods for the synthesis of polymer arrays (e.g., arrays of DNAs).Suppose one wants to produce 50,000 chips per year with each chip having60-mers of the appropriate four nucleotide bases at each well(electrode). To make such a chip, one would need as many as 60×4bases=240 base addition cycles to synthesize the desired completedpolymers. Assume each base addition cycle takes 15 minutes. So it wouldtake 240×15=3,600 min (60 hrs) or 2.5 days to manufacture a chip havingthe functionalized polymers on the chip, and each chip functionalizinginstrument could produce 365/2.5=146 chips per year. Thus, one wouldneed about 350 functionalizing instruments working for 24 hours a dayand 7 days a week to produce 50,000 chips. Assume that a functionalizinginstrument costs about $150,000 to operate per year, including purchaseprice the cost of consumed chemicals and salaries of people to run andrepair the instrument. In this case, the annual cost operating a factoryrunning 350 functionalizing instruments to produce 50,000 chips one at atime would cost about $52M. Thus, the cost per chip would be about$1,000.

The above example demonstrates that the functionalization methods toproduce ‘one chip at a time’ results in very high unit costs of thechips produced due to a lack of technology to integrate manufacturing ofmultiple dies in parallel on a single wafer. For many major applicationsof interest (e.g., disease diagnostics based on the application of largenumbers of DNA arrays), such a high unit cost per chip would likely tobe prohibitive. Other problems facing the ‘one chip at a time’manufacturing are between-chip variations in functionalization caused bysubtle differences between functionalizing instruments. The embodimentsof the invention reduce between-chip variations and address the demandto markedly reduce the chip manufacturing cost in order to realisticallyaccommodate the need for greater DNA array analysis by arriving at anovel wafer-scale electrochemical synthesis of polymer arrays.

Some of the features of the embodiments of this invention include: (1)individually addressable electrodes of each of the individual dies on awafer, the electrodes comprising an electrode to generate anelectrochemical reagent (preferably, an anode to generate an acid), aworking electrode to synthesize polymer and a confinement electrode toconfine protons or hydroxyl ions; and (2) parallel synthesis of polymers(DNA or peptides, for example) on the working electrodes of differentdies on a wafer by using an electrochemically generated deprotectingreagent (an acid, a base or a free radical, for example) that reactswith molecules on the working electrode surface to deprotect,polymerize, and/or protect the molecules using an acid-catalyzed polymersynthesis method.

One embodiment of the invention includes connecting the pads on the diesto the pads on the wafer through multilevel interconnects (two level ormore) fabricated across a scribe line to enable the parallel synthesisof polymers on the electrodes at multiple dies on a wafer.

Another embodiment of the invention for parallel addressing pads havingthe same functionality on the different dies includes via interconnectsthat traverses from the front side to the backside of the wafer. The viainterconnects could furthermore connected to connections points outsideof a wafer located in processing tool such as a wafer holder.

One embodiment of the invention is an apparatus to enable wafer levelsynthesis of polymers on the electrodes that includes microcells with awafer holder separated from a counter plate by a micro-gap, a fluiddelivery, pad contacts and control modules.

Another embodiment of the invention includes fabrication ofnano-material structures on the dies of the wafer in parallel byaddressing the electrodes having the same functionality in parallel andattaching functionalized nano-materials (such as DNA modifiedcomplementary nucleotide) to these electrodes by means of hybridizationand/or electrochemical attachment.

The embodiments of the invention could use silicon technology tofabricate interconnects for silicon chips to enable on-die synthesis ofpolymers such as DNA, peptides, and DNA-functionalized complementarynucleotide. Optionally, the embodiments of the invention could use waferprocessing cluster tools (process instruments) for synthesis. Typically,in volume silicon processing, a manufacturing line has a cluster ofinstruments (several identical instruments). Each can support a processstep or multiple process steps. By the embodiments of the invention,polymer synthesis can be treated as another process step in a devicemanufacturing line. A cluster of instruments can be configured within afacility to perform wafer level synthesis for efficient high volumemanufacturing.

In one embodiment of the present invention, polymers on a plurality ofdies on a wafer substrate are synthesized as follows. First, a terminalend of a monomer, nucleotide, or linker molecule (i.e., a molecule which“links,” for example, a monomer or nucleotide to a substrate) isprovided with at least one reactive functional group, which is protectedwith a protecting group removable by an electrochemically generatedreagent. The protecting group(s) is exposed to reagentselectrochemically generated at the electrode and removed from themonomer, nucleotide or linker molecule in a first selected region toexpose a reactive functional group. The substrate is then contacted withthe monomer or a pre-formed molecule (called the first molecule) suchthat the surface bonds with the exposed functional group(s) of themonomer or the pre-formed molecule. The first molecule may also bear atleast one protected chemical functional group removable by anelectrochemically generated reagent.

The monomer or pre-formed molecule can then be deprotected in the samemanner to yield a second reactive chemical functional group. A differentmonomer or pre-formed molecule (called the second molecule), which mayalso bear at least one protecting group removable by anelectrochemically generated reagent, is subsequently brought in thevicinity of the substrate to bond with the second exposed functionalgroup of the first molecule. Any unreacted functional group canoptionally be capped at any point during the synthesis process. Thedeprotection and bonding steps can be repeated sequentially at theplurality of the predefined regions on the substrate until polymers oroligonucleotides of a desired sequence and length are obtained.

In another embodiment of the present invention, polymers on a pluralityof dies on a wafer substrate are synthesized as follows. First, asubstrate of a wafer having one or more molecules bearing at least oneprotected chemical functional group bonded on an array of electrodes ona plurality of dies is obtained. The array of electrodes is contactedwith a buffering or scavenging solution. Following application of anelectric potential to selected electrodes in the array of electrodessufficient to generate electrochemical reagents capable of deprotectingthe protected chemical functional groups, molecules on the array ofelectrodes are deprotected to expose reactive functional groups, therebypreparing them for bonding. A monomer solution or a pre-formed molecule(called the first molecule), such as proteins, nucleic acids,polysaccharides, and porphyrins, is then contacted with the substratesurface of the wafer and the monomers or pre-formed molecules are bondedin parallel with a plurality of deprotected chemical functional groupson a plurality of dies on the wafer.

Another sufficient potential is subsequently applied to selectelectrodes in the array to deprotect at least one chemical functionalgroup on the bonded molecule or another of the molecules bearing atleast one protected chemical functional group on a plurality of dies onthe wafer. A different monomer or pre-formed molecule (called the secondmolecule) having at least one protected chemical functional group issubsequently bonded to a deprotected chemical functional group of thebonded molecule or the other deprotected molecule located at a pluralityof dies of the wafer. The selective deprotection and bonding steps canbe repeated sequentially until polymers or oligonucleotides of a desiredsequence and length are obtained. The selective deprotection step isrepeated by applying another potential sufficient to effect deprotectionof a chemical functional group on a bonded protected monomer or a bondedprotected molecule. The subsequent bonding of an additional monomer orpre-formed molecule to the deprotected chemical functional group(s)until at least two separate polymers or oligonucleotides of desiredlength are formed on the substrate.

In order to produce separate and pure polymers and to minimize, and, ifpossible to eliminate, chemical cross-talk between spots of a die orbetween dies on a wafer, it is desirable to keep the reactive molecules(e.g., protons, hydroxyl ions, or free radicals) confined to the areaimmediately proximate to a spot. Preferred embodiments of the presentinvention use a confinement electrode to confine reactive molecules, andthat actively prevents chemical cross-talk caused by diffusion of theelectrochemically generated ions from one spot to another spot within adie or between dies. For example, when an electrode exposed to anaqueous or partially aqueous media is biased to a sufficiently positive(or negative) potential, protons (or hydroxyl ions) may be produced asproducts of water hydrolysis. Protons, for example, are useful forremoving electrochemical protecting groups from several molecules usefulin combinatorial synthesis, for example, peptides, nucleic acids, andpolysaccharides.

The embodiments of the invention can be used to carry out theelectrochemical syntheses of polymers such as DNA and peptides accordingto any of a variety of approaches known to person skilled in the art.For example, any of a variety of reduction/oxidation (redox) reactionsmay be employed to electrochemically control the localization and pH ofa solution on Si-based electrodes to enable the attachment andelongation of polymers. In such methods, the electrical current drivesthe oxidation of an appropriate molecule at the anode(s) and thereduction of another molecule at the cathode(s) to control the kineticsand stoichiometry of acid-catalyzed organic syntheses on a Si-basedcircuit Such methods can also be used to generate high pH (basic)solutions, and to drive any other electrochemical redox reactions knownto one skilled in the art that may or may not result in pH changes(e.g., can also be used to generate reactive free radicals).

In one embodiment of the invention, the electrochemically generated acidsolutions also can enable coupling reactions between the monomers andacceptors that are widely used to synthesize polymers such as nucleicacid oligomers and peptides.

Another embodiment of the invention is electrochemical detection usingthe array chip. For example, a variety of published methods describe DNAbinding detection. Typically these methods employ measurements ofcurrent flow across a DNA monolayer tethered to a circuit on a siliconsubstrate. Current flow properties proportionately change when the DNAmonolayers are bound by an appropriate redox molecule-tagged test DNA oruntagged DNA that is co-added with a redox-active molecule thatspecifically binds double stranded DNA. Enzyme amplification methods canalso be incorporated into such assays in order to enhance theelectrochemical signal generated by binding events. Note that thesemethods can also be adapted by one skilled in the art to measure thebinding between other molecular species such as between two proteins ora protein and a small molecule.

The embodiments of the invention cover arrays used for research(typically high density with many different probes) as well asDiagnostics (typically low density because one is generally looking foronly a few specific analytes in a sample for diagnostics). The arraychip of an embodiment of the invention could be uses for screening largenumbers of polymers for biological activity, for example. To screen forbiological activity, for example, in the field of pharmaceutical drugdiscovery, the substrate is exposed to one or more receptors such asantibodies, whole cells, receptors on vesicles, lipids, or any one of avariety of other receptors. The probes are preferably labeled with, forexample, an electrochemical marker, an electro-chemiluminescent marker,a chemiluminescent marker, a fluorescent marker, a radioactive marker,or a labeled antibody reactive with the probe. The position of the probeon the substrate after hybridization is detected with, for example,electrochemical, fluorescence or autoradiographic techniques.

The array chip could also be used for therapeutic materials development,i.e., for drug development and for biomaterial studies, as well as forbiomedical research, analytical chemistry, high throughput compoundscreening, and bioprocess monitoring. An exemplary application includesapplications in which various known ligands for particular receptors canbe placed on the array chip and hybridization could be performed betweenthe ligands and labeled receptors.

Yet another application of the array chip of an embodiment of thisinvention includes, for example, sequencing genomic DNA by the techniqueof sequencing by hybridization. Non-biological applications are alsocontemplated, and include the production of organic materials withvarying levels of doping for use, for example, in semiconductor devices.Other examples of non-biological uses include anticorrosives,antifoulants, and paints.

It is specifically contemplated that the array chip and/or the methodsof manufacturing the array chip of an embodiment of the invention couldbe used for developing new materials, particularly nano-materials formany purposes including, but not limited to corrosion resistance,battery energy storage, electroplating, low voltage phosphorescence,bone graft compatibility, resisting fouling by marine organisms,superconductivity, epitaxial lattice matching, or chemical catalysis.Materials for these or other utilities may be formed proximate to one ora plurality of the electrodes in parallel on a plurality of dies of asilicon wafer, for example. Alternatively, materials may be formed bymodifying the surface of one or a plurality of electrodes on a pluralityof dies by generating reagents electrochemically.

It is further contemplated that an array chip of the embodiments of theinvention could be used to develop screening methods for testingmaterials. That is, reagents electrochemically generated by an electrodeon a die could be used to test the physical and chemical properties ofmaterials proximate to the electrode. For example, the array chip couldbe used for testing corrosion resistance, electroplating efficiency,chemical kinetics, superconductivity, electro-chemiluminescence andcatalyst lifetimes.

The advantageous characteristics of some of the embodiments of theinvention are illustrated in the following examples, which are intendedto be merely exemplary of the invention.

The array chips of the embodiments of the invention are preferablysilicon bio-chips built by using silicon process technology and SRAMlike architecture with circuitries including electrode arrays, decoders,serial-peripheral interface, on chip amplification, for example. Thesilicon bio-chip for wafer level polymer synthesis could be fabricatedby the following steps, for example, which are described with referencewith FIG. 1.

First, fabricate a wafer shown in FIG. 1( a) containing a CMOS circuitryto be connected to individually addressable electrodes. The CMOScircuitry amplifies the signal, reads and writes information on theindividually addressable electrodes (i.e. functionalize the individuallyaddressable electrodes and read out binding events), besides performingother functions.

Second, fabricate the individually addressable electrodes for eachindividual dies on the wafer including anodes to generate acid, workingelectrodes to synthesize polymer and confinement electrodes to confineprotons. These electrodes are shown in FIG. 1( b).

Third, interconnect the die pads on the dies on the wafer to wafer levelpads for parallel synthesis of polymers on the working electrodes. Thedie pads are shown as DP1, DP2, DP3 and the wafer-pads are shown as WP1,WP2, WP3 in FIG. 1( a). The die pads are used for power, signaldelivery, for example. The die pads could be interconnected by eitherusing a multilevel interconnect (two or more layers) accoss a scribeline on the front side of the wafer as shown in FIG. 1( b) or using avia interconnect that traverses from the front side of the wafer to thebackside of the wafer FIG. 1( c). The via connect could be connected toelectrical contact on the backside of the wafer and further to anoutside contact located outside the wafer in a processing tool such as awafer holder. FIG. 1( c) shows a localized and generalized cross-sectionview of the through wafer interconnect concept. The diagram shows theactive electrode in an ILD well, the CMOS circuitry that controls theelectrode, and the IO (input/output interconnect to the outsideinstrument) connecting the CMOS circuitry to pads on the backside of thewafer. Electrochemical synthesis occurs on the functionalized electrodeswhen activated. The CMOS circuitry, shown here as a block diagram,decodes the input address and signal. In contract to typical silicondevices in which power and signal is input to the CMOS circuitry througha topside wire bond pad or solder bump connection, in this technique,the connection is made through the backside via.

Fourth, perform parallel synthesis of a material such as polymers (e.g.,DNA) or a nano-material on the individually addressable workingelectrodes of different spots of a die or of different dies on the waferby using electrochemically generated reagents (acids, bases, radicals,etc.) that reacts with a molecule on the surface of the workingelectrode. FIG. 2 shows CMOS switching scheme for individuallyaddressing different working electrodes on the wafer. In FIG. 2, eachdie pad on the die branches into a large array of synthesis electrodes.These electrodes are exposed to the electrolyte and can be modified withpolymer (e.g., DNA). CMOS switches ensure that a given electrode (or anentire column, or an entire row) can be modified one base pair at atime. Voltage source and counter electrode (plating tool) are shown tocomplete the electrical circuit. FIG. 3 shows an exemplary scheme forthe synthesis of polymers on the individually addressable electrodes onthe wafer by generating acid on the electrodes through anelectrochemical reaction to polymerize, protect, and/or deprotectpolymer subunits using an acid-catalyzed polymer synthesis scheme. Thesynthesis scheme of FIG. 3 is particularly applicable for acid catalyzedpolymer synthesis for the manufacture of nucleic acids such as DNA. FIG.3 shows that by applying a voltage to a metal electrode, protons aregenerated in a redox buffer. The protons in turn catalyze the synthesisof DNA one base pair at a time according to the synthesis scheme of FIG.3. While performing of the parallel synthesis of a material it would bepreferable to apply a specific potential to confinement electrodessurrounding the working electrodes to effectively confine protons to theworking electrodes by an electrochemical means and to preventdiffusion-controlled spreading of protons to neighboring workingelectrodes Another embodiment includes parallel synthesis of polymerssuch as DNA on the working or synthesis electrodes of different dies byconnecting to the pads on the wafer using a sealed probe card.

Preferably, the device making electrical connection from the instrumentcontrolling the synthesis should be isolated from the reagents washingover the wafer. If the connecting pads on the wafer are sufficiently farfrom the electrodes, this is accomplished with a single wafer-level sealor gasket. The backside via technique and the wafer-level pad technique(with die connected with the inter-die metal lines) are examples ofmaking electrical connection the instrument controlling the synthesis tothe dies by embodiments of the invention. A single gasket could encircleall the electrodes and the connections/pads to the instrument could beoutside the gasket on the wafer edge or wafer backside. It is alsopossible to isolate the electrical connections independently and locallywith a probe card that lands on each pad on each die. In this case,there would preferably be no need to connect pads between die or routeconnection of the pads to the backside. For example, if the wafer has100 die with 100 pads each, a probe card with 100×100=10000 connectingprobes could land on each pad on the wafer, isolate that connection fromthe reagents with a pad-level gasket, and perform the synthesis. Waferpads could employ seal probe cards or a wafer level isolating gasket.

Yet, in another embodiment related to the fabrication of nano-materialstructures on the dies of the wafer in parallel could be performed byaddressing in parallel the working electrodes and attachingfunctionalized nano-materials (such a DNA modified complementarynucleotide) to this electrodes by means of hybridization and/orelectrochemical attachment.

Fifth, dice and cut the wafer into individual dies.

Finally, mount the die on a substrate through the use of the scribelines on the front side of the wafer or through the use of contacts onthe backside of the wafer.

This application discloses several numerical range limitations thatsupport any range within the disclosed numerical ranges even though aprecise range limitation is not stated verbatim in the specificationbecause the embodiments of the invention could be practiced throughoutthe disclosed numerical ranges. Finally, the entire disclosure of thepatents and publications referred in this application, if any, arehereby incorporated herein in entirety by reference.

1. A wafer comprising a plurality of dies and interconnects, wherein theplurality of dies are integral portions of the wafer and theinterconnects are on the wafer, wherein an individual die comprises aworking electrode and, a die pad, an electrode operable to generate adeprotecting reagent, and a confinement electrode surrounding theworking electrode and the electrode operable to generate a deprotectingreagent to confine a reductive molecule or an oxidative molecule and adie pad on the wafer, wherein the die pads of the plurality of dies asintegrated portions of the wafer are interconnected by the interconnectsto carry out parallel chemical reactions on a plurality of workingelectrodes of the plurality of dies as integrated portions of the wafer,wherein each of said plurality of dies comprises a surface disposed tobe exposed to a test solution, wherein each of said plurality of diescomprises electrical interconnects adaptable to electricallyinterconnect with electrical connections provided on a substrate, andwherein an individual die diced from the wafer is electrically operablewhen mounted on said substrate.
 2. The wafer of claim 1, wherein thechemical reaction is an electrochemical redox reaction.
 3. The wafer ofclaim 1, wherein die pads of the plurality of dies are interconnected onthe wafer to carry out a chemical reaction in parallel on the pluralityof working electrodes.
 4. The wafer of claim 1, wherein the reductivemolecule or the oxidative molecule is a proton, a hydroxyl ion, a freeradical, or combination thereof.
 5. The wafer of claim 1, wherein theelectrode to generate a deprotecting reagent is an anode.
 6. The waferof claim 1, further comprising a scribe line on the wafer and the diepads of the plurality of dies are interconnected across the scribe line.7. The wafer of claim 6, wherein the die pads of the plurality of diesare interconnected across the scribe line on a front side of the waferpad of the wafer.
 8. The wafer of claim 6, wherein the die pads of theplurality of dies are interconnected to the wafer pad through two ormore level interconnects across the scribe line.
 9. The wafer of claim1, wherein the wafer comprises a via interconnection through the wafer,and the die pads of the plurality of dies are interconnected by the viainterconnection.
 10. The wafer of claim 9, wherein the die pads of theplurality of dies are interconnected to a connection outside the waferthrough the via interconnect.
 11. The wafer of claim 1, wherein the diepads of the plurality of dies are interconnected by a probe card. 12.The wafer of claim 1, further comprising a CMOS switch between the diepad and the working electrode.
 13. The wafer of claim 1, wherein thedeprotecting reagent is an acid, a base or a free radical.
 14. The waferof claim 1, wherein the wafer is a silicon substrate.
 15. The wafer ofclaim 1, wherein the wafer comprises a plurality of targets.
 16. Thewafer of claim 1, wherein the wafer comprises a cDNA target or anoligonucleotide target.
 17. The wafer of claim 1, wherein the pluralityof dies are demarcated by scribe lines on a front side of the wafer andthe die pads are interconnected by multilevel interconnects across thescribe lines.
 18. A wafer comprising a plurality of dies andinterconnects, wherein the plurality of dies are integral portions ofthe wafer and the interconnects are on the wafer, wherein an individualdie comprises a working electrode, a die pad, an electrode operable togenerate a deprotecting reagent, and a confinement electrode surroundingthe working electrode and the electrode operable to generate adeprotecting reagent to confine a reductive molecule or an oxidativemolecule and a die pad on the wafer, wherein the die pads of theplurality of dies are interconnected as integrated portions of the waferby the interconnects to carry out parallel chemical reactions on aplurality of working electrodes of the plurality of dies as integratedportions of the wafer, wherein each of said plurality of dies comprisesa surface disposed to be exposed to a test solution, wherein each ofsaid plurality of dies comprises electrical interconnects adaptable toelectrically interconnect with electrical connections provided on asubstrate, and wherein the wafer is configured to be diced such that theplurality of dies are electrically operable when diced from the wafer.19. The wafer of claim 18, wherein the chemical reaction is anelectrochemical redox reaction.
 20. The wafer of claim 18, wherein diepads of the plurality of dies are interconnected on the wafer to carryout a chemical reaction in parallel on the plurality of workingelectrodes.
 21. The wafer of claim 18, wherein the reductive molecule orthe oxidative molecule is a proton, a hydroxyl ion, a free radical, orcombination thereof.
 22. The wafer of claim 18, wherein the electrode togenerate a deprotecting reagent is an anode.
 23. The wafer of claim 18,further comprising a scribe line on the wafer and the die pads of theplurality of dies are interconnected across the scribe line.
 24. Thewafer of claim 23, wherein the die pads of the plurality of dies areinterconnected across the scribe line on a front side of the wafer to awafer pad of the wafer.
 25. The wafer of claim 23, wherein the die padsof the plurality of dies are interconnected to the wafer pad through twoor more level interconnects across the scribe line.
 26. The wafer ofclaim 18, wherein the wafer comprises a via interconnection through thewafer, and the die pads of the plurality of dies are interconnected bythe via interconnection.
 27. The wafer of claim 26, wherein the die padsof the plurality of dies are interconnected to a connection outside thewafer through the via interconnect.
 28. The wafer of claim 18, whereinthe die pads of the plurality of dies are interconnected by a probecard.
 29. The wafer of claim 18, further comprising a CMOS switchbetween the die pad and the working electrode.
 30. The wafer of claim18, wherein the deprotecting reagent is an acid, a base or a freeradical.
 31. The wafer of claim 18, wherein the wafer is a siliconsubstrate.
 32. The wafer of claim 18, wherein the wafer comprises aplurality of targets.
 33. The wafer of claim 18, wherein the wafercomprises a cDNA target or an oligonucleotide target.
 34. The wafer ofclaim 18, wherein the plurality of dies are demarcated by scribe lineson a front side of the wafer and the die pads are interconnected bymultilevel interconnects across the scribe lines.